In the year of 2013,using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax LT (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389), and human transforming growth factor alpha (TGF). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant approach to obtaining high levels of lipopolysaccharide-free recombinant protein. During 2013 we also deleted additional secreted proteases from B. anthracis. The strain BH480, created in 2012, and deleted of eight proteases, has proven useful in the preparation of a number of recombinant proteins. Improved Cre-loxP and Flp-FRT gene knockout systems were used to sequentially delete the genes encoding the eight proteases (InhA1, InhA2, camelysin, TasA, NprB, MmpZ, CysP1 and VpR). For general use as a host for the production of recombinant proteins, the strain was cured of pXO1 and made permanently sporulation deficient. In 2013, we analyzed culture supernatants of strain BH480 by mass spectrometry (MS) to identify extracellular proteases that continue to be produced at trace levels that might degrade valuable endogenous proteins (anthrax toxin proteins that are vaccine candidates) or heterologous proteins. One additional secreted protease was identified: GBAA_2183 neutral metalloprotease NprC. This protease was genetically deleted, producing strain BH490, which lacks 9 proteases. The improved Saccharomyces cerevisiae Flp-FRT recombinase system was used in B. anthracis for deletion of the nprC gene. The Flp-FRT method replaced the nprC gene with 48-bp FRTsequences as a result of action of the cloned Flp recombinase. Insertion of the FRT sequence avoided potential problems associated with the presence of loxP sites remaining in BH460 due to deletion of other proteases. BH490 is therefore recommended as a new host strain for recombinant protein production. Also, using newly developed technology for DNA insertion into the genome of the B. anthracis, we replaced the nprC gene with atxA, producing strain BH490A, a new approach that may provide a basis for a new B. anthracis host/vector system. In the year of 2013, we also used our gene targeting tools to characterize the function of the B. anthracis atxA gene. Using Next Generation sequencing, we found 15 pXO1-encoded genes and 3 chromosomal genes that were strongly regulated by the separate or synergistic actions of AtxA and carbon dioxide. The majority of the regulated genes responded to both AtxA and carbon dioxide rather than to just one of these factors. Interestingly, we identified two previously unrecognized small RNAs that are highly expressed under physiological carbon dioxide concentrations in an AtxA-dependent manner. Expression levels of the two small RNAs were found to be higher than that of any other gene differentially expressed in response to these conditions. Secondary structure and small RNA-mRNA binding predictions for the two small RNAs suggest that they may perform important functions in regulating B. anthracis virulence. The components of the AtxA regulatory pathway that we identified and their interactions with CO2 represent molecular targets for specific inhibitory interventions aimed at decreasing the pathogenesis of B. anthracis. In a fourth project executed in 2013, we analyzed genes and sequences that are needed for maintenance of the key virulence plasmid pXO1, which encodes all three anthrax toxin proteins. In previous work we identified and isolated the region of pXO1 that is responsible for plasmid replication, and thereby defined a minireplicon that was different from one reported by others. It has now become clear through the work of others that the minireplicon we defined is universally conserved among the large family of similar bacillus species plasmids, validating our identification. However, DNA replication alone does not assure that every daughter cell receives a copy of pXO1 when cell division occurs. Thus, we sought to understand the mechanism that causes accurate segregation of plasmids between daughter cells, an active process that assures plasmid maintenance in the bacterial population. We applied the new S. cerevisiae Flp-FRT recombination system to the genetic manipulation of B. anthracis. Using this system along with the Cre-loxP system, we identified two regions of pXO1 that are each able to cause plasmid retention. One of these regions lies within pXO1 ORFs 0069-0084. This region is different from the maintenance system reported previously for the large pBtoxis in B. thuringiensis, which has some similarities to pXO1. This knowledge will provide targets for anti-infective agents those that do not directly kill the pathogen but instead render it less virulent, thereby allowing host immune responses to effectively combat it.